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Definitions

• the invention relates to the therapy of Duchenne Muscular Dystrophy (DMD) by means of modulating the amount of a specific miRNA.
• DMD Duchenne Muscular Dystrophy
• DAPC Dystrophin- Associated Protein Complex
• exon skipping allows the rescue of dystrophin synthesis through the production of a shorter but functional mRNA (Denti et al., 2006, 2008).
• the authors identify a miRNA (miR-31), particularly abundant in dystrophic regenerating fibers that represses dystrophin expression. They also show that by means of sequestering miR-31 or of protecting its target sequence on the dystrophin 3'UTR, an increase of translation of dystrophin is obtained.
• miRNAs are molecules known to play crucial functions in the differentiation commitment of several cell types and to be involved in many pathological processes.
• the authors identified a specific signature of miRNAs that is correlated with the DMD pathology. They found a different miRNA expression profile between wild type and Duchenne conditions.
• a specific miRNA, miR-31 was found at higher levels in mdx as well as in human DMD muscles with respect to wild type conditions.
• miR-31 increases by 30-50 fold in mouse dystrophic muscles and 7-10 fold in human DMD biopsies.
• nucleic acid molecules act as a "sponge" for miR-31.
• they contructed a molecule having a RNA sequence containing multiple target sites for miR-31.
• the sequence of such molecules is included in the 3'UTR of a carrier mRNA and is specifically expressed under the control of a muscle-specific promoter.
• nucleic acid molecules act as "protectors" of dystrophin mRNA against miR-31 repression, achieving the same result of functional inactivation of the action of the miR-31 miRNA on the dystrophin RNA.
• they constructed nucleic acid molecules comprising a region complementary to the 3'UTR dystrophin mRNA region which is recognized by the miR-31 sequence (5'- GGCAAG-3'). By competing for miR-31 binding, such molecules are able to prevent the trans lational repression mediated by miR-31.
• the molecule is provided as a synthetic oligo or as part of a chimeric RNA in a gene therapy approach.
• the protector sequence i.e.
• nucleotides for efficient uptake in vivo, would preferably have the length of approximately 15 nucleotides (5 '-GAAAUGGCAAGUUAU-3 ' ; SEQ ID No. 1), while, i.e. for in vitro transfection, could be longer up to appr. 30 nucleotides, as for example, the 23 oligonucleotide 5'- CC AUAUAAAGAAAUGGC AAGUUA-3 ' ; SEQ ID No. 2. The region corresponding to the miR-31 sequence recognizing the dystrophin 3 'UTR is underlined.
• nucleic acid molecule or a derivative thereof able to functional inactivate the action of the miR-31 miRNA on the dystrophin RNA (NCBI accession ID: NM 004006).
• a "derivative" is a nucleic acid molecule, as a DNA molecule, coding the nucleic acid molecule of the invention, or a nucleic acid molecule comprising the nucleotide sequence of the nucleic acid molecule of the invention.
• the molecule is able to bind to the miR-31 by sequence complementarity and consequently selectively sequester the miR-31 in muscle cells.
• the sequence preferably comprises more than one sequence, each one being complementary to the miR-31 sequence: 5 '- aggcaagaugcuggcauagcu-3'(SEQ ID No. 3).
• the miRNA in order not to get the cleavage of the substrate (the miRNA), a non perfect match is preferred (Rivas FV et al. 2005); therefore at least one nucleotide of the nucleic acid molecule of the invention is not complementary to the corresponding nucleotide comprised in the region from nt. 9 to nt. 14 of the miR-31 sequence.
• nucleic acid molecule of the invention are not complementary to the corresponding nucleotides comprised in the region from nt. 9 to nt. 14 of the miR-31 sequence.
• nucleic acid molecule essentially consists of a molecule having the following sequence: 5 ' ggcagcuauguuugcaucuugccucacagcuauguuugcaucuugccucacagcuauguuugcaucuugcc ucacacagcuauguuugcaucuugccuccgc-3'(SEQ ID No. 4).
• the nucleic acid molecule or a derivative thereof as above disclosed may be advantageously comprised in the 3 'UTR of a transcript whose expression is driven by a muscle-specific promoter.
• an expression vector for gene therapy comprising a sequence encoding the nucleic acid molecule fused to the a muscle-specific promoter.
• a muscle-specific promoter is a promoter driving selectively the transcription of an operatively linked sequence in muscle cell lineages.
• the expression vector for gene therapy is preferentially an AAV (Adeno-associated viruses) vector comprising a muscle- specific promoter.
• the muscle-specific promoter is active in early phases of muscle differentiation.
• the expression vector for gene therapy is advantageously produced as AAV viral particle which preferentially transduces muscle cells.
• the molecule is able to compete with the miR-31 molecule for the binding to the 3'UTR dystrophin mRN A.
• the molecule comprises a sequence that is complementary to the 3 'UTR dystrophin mRNA region which is recognized by the miR-31 sequence 5'-GGCAAG-3'.
• the nucleic acid molecule comprises a sequence that is complementary to the 3'UTR dystrophin mRNA region, said region comprising the sequence: 5'-CUUGCC-3'.
• the nucleic acid molecule is an oligonucleotide of appr. 15 nucleotides fully complementary to the 3'UTR dystrophin mRNA region comprising the sequence: 5 '- CUUGCC-3'; most preferably the nucleic acid molecule comprises the sequence 5'- GAAAUGGC AAGUUAU-3 ' (SEQ ID No. 1).
• the nucleic acid molecule is a modified synthetic oligonucleotide, preferably belonging to the group of: LNA (Locked Nucleic Acid), methylated oligos, phosphoro-thiolated oligos.
• the nucleic acid molecules of the invention as above disclosed that are able to functional inactivate the action of the miR-31 miRNA on the dystrophin RNA are also provided as an expression vector for gene therapy comprising a sequence encoding the nucleic acid molecule fused to the a muscle-specific promoter.
• a muscle-specific promoter is a promoter driving selectively the transcription of an operatively linked sequence in muscle cell lineages.
• the expression vector for gene therapy is preferentially an AAV vector comprising a muscle-specific promoter.
• the muscle-specific promoter is active in early phases of muscle differentiation.
• the expression vector for gene therapy is advantageously produced as AAV viral particle which preferentially transduces muscle cells.
• the combination of these two features namely the muscle specificity of the promoter and the selectivity for muscle cells of the viral particle, ensures the restricted expression of the nucleic acid molecule of the invention in muscle cells. It is a further object of the invention to provide the nucleic acid molecule as above disclosed for medical use, in particular for medical use for muscle degenerative disorders, as Muscular Dystrophies, particularly Duchenne Muscular Dystrophy.
• the nucleic acid molecule disclosed herein may be advantageously used for increasing the translation of dystrophin in a host in need thereof, by administering to the host a therapeutically active amount of the said nucleic acid molecule.
• a particular aspect is to improve the so-called exon-skipping therapeutic approach or other strategies where one wants to increase the efficiency of dystrophin translation in the treatment of Duchenne Muscular Dystrophy, to recover a correct dystrophin expression by administering the nucleic acid molecule of the invention, either directly or by means of gene therapy.
• FIG. 1 miR-31 expression.
• A miR-31 relative expression in WT and mdx mice, measured by qRT-PCR.
• B In situ hybridization of miR-31 in mdx gastrocnemius.
• C miR-31 relative expression in WT (black bars) and mdx (grey bars) mouse satellite cells, measured by qRT-PCR in growth medium (/) and at the indicated time points after shift to differentiation medium.
• D The same cells were analyzed by Western blot for dystrophin and actinin proteins.
• E qRT-PCR of miR-31 relative expression in human muscle biopsies from healthy (Ctrl), Duchenne (DMD) and Becker (BMD) donors.
• FIG. 1 qRT-PCR of miR-31 relative expression in human primary myoblasts from healthy donors (Ctrl, black bars) or DMD patients (DMD, grey bars) in growth medium (/) and at the indicated time points after shift to differentiation medium.
• G The same cells were analyzed by Western blot for Pax7, MHC, dystrophin and actinin proteins.
• A Northern and Western blot analysis on RNA (miR-31) and proteins (Dystrophin and actinin) from C2 mouse myoblasts in growth medium (GM) and at 3 and 5 days in differentiation medium (DM).
• FIG. 1 Representative Western blots with anti-dystrophin and anti-actinin antibodies in C2 myoblasts infected with a mock lentivirus (ctrl) or with one carrying a miR-31 expression cassette (LmiR-31) at 3 and 5 days of differentiation. In all the experiments the dystrophin levels were measured from three independent experiments and normalized to actinin expression (histograms at the bottom). miR-31 expression levels (panels miR-31) were measured by Northern blot.
• C As in panel B) with the difference that C2 myoblasts were transfected with an anti-miR-31 (LNA-31) or scrambled LNA oligos and incubated 3 days in differentiation medium.
• Dystrophin is target of miR-31.
• A Schematic representation of the constructs utilized: DMD-WT and DMD-mut contain dystrophin wild type 3 'UTR or its derivative mutant for the miR-31 target site respectively. Constructs miR-31s and Ctrl contain a 3 'UTR with and without 4 miR-31 target sites respectively.
• B C2 myoblasts were transfected with either a control luciferase (Ctrl) or DMD-WT and DMD-mut constructs.
• luciferase reporter plasmids were performed with a vector expressing miR-31 (pmiR-31) or with a control plasmid (pCtrl); luciferase activity measured after 24h.
• C Luciferase activity of the DMD-WT was measured in C2 myoblasts 24h after co- transfection with miR-3 Is or with a control vector.
• FIG. 4 Dystrophin miR-31 target protection.
• the DMD-WT construct was transfected in proliferating C2 myoblasts in combination with the pmiR-31 plasmid (bar#l, 2 and 3). Subsequently, LNA oligos complementary to the miR-31 site were transfected (bar#2) in parallel with control scrambled oligos sequence (bar#3). Luciferase activity was measured 36 hours after trans fection.
• FIG. 5 Inhibition of miR-31 activity enhances dystrophin rescue upon exon skipping.
• A Schematic representation of the exon skipping strategy in the human DMD D48-50 deletion.
• B D48-50 myoblasts were infected with a lentiviral construct expressing the antisense molecules able to induce exon skipping (Ul #51), alone (Ctrl) or together with a lentivirus expressing the sponge construct (miR-3 Is). Mock are uninfected D48-50 myoblasts.
• RQ Relative Quantification
• miR-31 target protector LNA oligos were locally injected in the right tibialis (+), while control scrambled oligos were administered in the contralateral leg (-). After an additional week, muscles were dissected and proteins analyzed. The diagram shows the average values of dystrophin accumulation from three independent experiments.
• Mature miRNAs as below show perfect sequence conservation between human and mouse.
• the mature sequence of the human miRNA miR-31 is:
• 5' aggcaagaugcuggcauagcu 3' (SEQ ID No. 3; Sanger ID No. MIMAT0000089, http://www.mirbase.org/); the underlined nucleotides refer to a sequence complementary to the 5 '-CUUGCC-3 ' sequence on the 3 'UTR of the Dystrophin RNA.
• the underlined sequence refers to nucleotides complementary to the 5'-CUUGCC-3' sequence on the 3'UTR of the Dystrophin RNA; the protector is able to compete for the binding of miR-31 to the same.
• RNA Preparation and Analysis Total RNA was prepared from liquid nitrogen powdered tissue homogenized in TRIzol reagent (Invitrogen). miRNAs analysis was performed using specific TaqMan microRNA assays (Applied Biosystems). Relative quantification was performed using sno55 as endogenous control for murine samples and U6 for human samples. Data were expressed as means ⁇ SEM, unless otherwise stated. Statistical significance of the differences between means was assessed by t-test (nonparametric). A probability of ⁇ 5% was considered significant.
• Muscle homeostasis depends on the concerted action of molecular mechanisms controlling myogenic proliferation, differentiation and maturation. Modulation of such processes occurs through the combined activity of transcriptional factors and miRNAs which control the expression of a complex network of target genes.
• muscle degenerative disorders such as Duchenne Muscular Dystrophy (DMD)
• muscle fiber breakage and degeneration is accompanied by a complex series of events including inflammatory infiltration, intense fibrosis and, most importantly, the activation of satellite cells which provide supply for new tissue formation.
• microRNA-mediated control of gene expression appears to be especially important in muscle differentiation (Naguibneva et al., 2006; Chen et al., 2006) and in muscle degenerative diseases where their expression was described to be strongly deregulated (Eisenberg et al., 2007).
• mdx mice Profile analysis in Duchenne versus wild type muscles indicated that several classes of miRNAs are differentially expressed in mdx mice (Greco et al., 2009). Among these, we found that in a two-month old mdx muscle, miR-31 displays a 40-fold enrichment with respect to control muscles (Fig. IA). Due to such conspicuous abundance and in consideration of the fact that dystrophic muscles undergo intense tissue reorganization with massive degeneration and regeneration, we tested miR-31 localization by in situ hybridization. Figure I B shows that miR-31 has a preferential localization in activated/differentiating satellite cells recognized by the characteristic phenotype of mononucleated fibers, highly abundant in mdx conditions.
• miR-31 was observed to be highly abundant also in human DMD biopsies when compared to wild type and Becker muscles (Fig. IE).
• DMD myoblasts appear to have increased regenerative and lower differentiation potential than control cells as shown by the expression of the Pax7 regenerative factor (Buckingham and Relaix, 2007) in growth conditions and by the delayed appearence of the myosin heavy chain (MHC) protein after shift to differentiation conditions (Fig. IG).
• the Pax7 detection in primary DMD myoblasts is likely to be due to the fact that, similarly to mdx muscles, DMD biopsies include a relevant proportion of activated satellite cells as expected in a dystrophic muscle.
• Fig. 2B When cells were induced to differentiate, a consistent decrease of dystrophin (more than 2-fold) was observed in the presence of persistent expression of miR-31 at 3 days (Fig. 2B); on the contrary, increase in dystrophin levels was detected when cells were treated with anti-miR-31 LNAs along 3 days of differentiation (Fig. 2C).
• the limited increase (40%) of dystrophin synthesis when cells are treated with anti-miR-31 LNA is probably due to the fact that at that stage of differentiation miR-31 levels start to decrease (Fig. 2A). No effects were observed with a plasmid expressing an unrelated miRNA (lanes Ctrl) or with scrambled LNA oligos (lane scramble).
• Dystrophin mRNA was validated as a miR-31 target through the classical luciferase assay: wild type and mutated derivatives of the dystrophin 3'UTR were fused to the luciferase reporter ORF (Fig. 3A) and enzyme activity was measured in conditions of miR-31 overexpression (Fig. 3B). The results indicate that miR-31 represses luciferase activity only in the presence of a dystrophin wild type 3'UTR (DMD-WT). When the same cells were treated with a Sponge construct (Gentner et al, 2009; Brown and Naldini, 2009) containing multiple sites for miR-31 (Fig. 3 A, miR-3 Is), a partial recovery of luciferase activity was obtained (Fig. 3C).
• miR-31 protector contains sequences complementary to the miR- 31 target site present on the dystrophin 3'UTR.
• C2 myoblasts were transfected with the construct containing the dystrophin 3'UTR fused to the luciferase ORF (DMD-WT) together with a plasmid overexpressing miR-31. Under these conditions, the luciferase activity is strongly inhibited due to the repressing activity of miR-31.
• a LNA oligonucleotide complementary to the recognition site of miR-31 was co-transfected, luciferase activity was strongly increased (Fig. 4, bar#2).
• no treatments (bar#l) or scrambled LNA oligos treatments (bar#3) were not able to interfere with miR-31 repressing activity.
• Fig. 5A One of the most utilized approaches for the DMD gene therapy is exon skipping (Fig. 5A).
• This methodology makes use of antisense molecules able to induce the exclusion, during the splicing reaction, of specific dystrophin mutated exons, producing a shorter but functional RNA (De Angelis et al., 2002, Aartsma-Rus et al., 2009).
• dystrophin rescue can be obtained with consequent partial recovery of the pathogenic traits and rescue of morpho-functional parameters (Goyenvalle et al., 2004, Denti et al., 2006 and 2008, Aartsma-Rus et al., 2009).
• DMD myoblasts deriving from a patient with deletion of dystrophin exons 48-50 were infected with a lentiviral construct expressing an antisense RNA able to induce skipping of exon 51 (lenti-Ul#51). Exclusion of this exon from the mature mRNA is able to rescue the open reading frame of the transcript and to induce the production of a shorter but functional dystrophin (De Angelis et al., 2002). However, through this strategy, the levels of dystrophin are rescued at maximum 10-20% of wild type levels.
• microRNA miR-181 targets the homeobox protein Hox- Al 1 during mammalian myoblast differentiation. Nat Cell Biol. ,8 :278-84.

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